Project Summary Powassan virus (POWV) is an emerging tick-borne flavivirus that when transmitted to humans through the bite of an infected tick causes symptoms ranging from asymptomatic infection to encephalitis and death. There is no specific treatment available to combat the disease. Little is known about the factors contributing to disease severity and the role of antibody responses in protection. The geographical distribution of POWV encephalitis cases overlaps with that of Lyme disease and both pathogens can be transmitted by infected Ixodes scapularis ticks. The primary goal of this proposal is to examine the characteristics of effective antibody responses to POWV through the identification and characterization of human monoclonal antibodies with potent neutralization capacity. In addition, the study aims to determine the prevalence of POWV exposure in Lyme disease patients to inform future studies on the possible consequences of dual infection. To achieve the goals, both an existing and a newly recruited cohort of patients with Lyme disease will be screened for serological evidence of POWV exposure. Serum from positive individuals will be tested in quantitative assays for neutralization potency. For this pilot project, antibodies recognizing the domain III of the POWV Envelope protein will be cloned from the memory B cells of those individuals with the most potent neutralization responses. The cloned antibodies will be characterized for their binding and neutralization properties by ELISA and reporter virus particle neutralization assays, using both POWV lineages and a panel of other flaviviruses. Future studies will discover antibodies against other epitopes of the virus. It is expected that highly neutralizing anti-POWV human monoclonal antibodies will be discovered that can be developed for possible therapeutic or diagnostic purposes. Overall, the information gained will also inform future vaccine design.